Phytochromes constitute one of the six well-characterized families of photosensory proteins in Nature. From the viewpoint of computational modeling, however, phytochromes have been the subject of much fewer studies than most other families of photosensory proteins, which is likely a consequence of relevant high-resolution structural data becoming available only in recent years. In this work, hybrid quantum mechanics/molecular mechanics (QM/MM) methods are used to calculate UV-vis absorption spectra of Deinococcus radiodurans bacteriophytochrome. We investigate how the choice of QM/MM methodology affects the resulting spectra and demonstrate that QM/MM methods can reproduce the experimental absorption maxima of both the Q and Soret bands with an accuracy of about 0.15 eV. Furthermore, we assess how the protein environment influences the intrinsic absorption of the bilin chromophore, with particular focus on the Q band underlying the primary photochemistry of phytochromes.

L'AChE, enzyme clé pour la neurotransmission, est la cible des neurotoxiques organophosphorés utilisés comme agents de guerre tels que le tabun. En inhibant irréversiblement l'AChE, ces neurotoxiques peuvent entraîner la mort par asphyxie. Des antidotes à ces organophosphorés existent : il s'agit d'oximes, qui sont des nucléophiles puissants. Malheureusement, aucune oxime à l'heure actuelle n'est capable de restaurer l'activité de l'AChE inhibée par le tabun. C'est pourquoi nous nous sommes particulièrement intéressés au complexe AChE-tabun en utilisant une méthodologie hybride QM/MM. Nous avons ainsi défini notre modèle théorique sur l'acétylcholine, substrat naturel de l'AChE, ce qui nous a permis de mettre en évidence la grande flexibilité de l'AChE et la difficulté de la traiter en chimie théorique quantitativement. Nous nous sommes ensuite penchés sur l'inhibition de l'AChE par le tabun pour montrer l'influence de la structure du tabun sur sa réactivité dans le site actif. Notamment le rôle stérique du groupement N-diméthyle a été souligné. Enfin, nous avons abordé la réactivation du complexe AChE-tabun. L'influence du mécanisme (une ou deux étapes) a permis d'expliquer la difficile réactivation du complexe AChE-tabun. Un nouveau réactivateur a pu être conçu à partir de ces informations, synthétisé et testé in vitro. Ce réactivateur n'est malheureusement pas efficace contre le complexe AChE-tabun du fait de paramètres physico-chimiques que nous ne pouvons pas prendre en compte d'un point de vue théorique. Notre travail a donc permis de mettre au point un modèle théorique efficace pour traiter les interactions de l'AChE avec différents substrats.

<p>The thesis contains two directions in the simulations of biomolecular systems. The first part (Chapter 2 - Chapter 4) mainly focuses on the simulations of electron transfer processes in condensed phase; the second part (Chapter 5 - Chapter 6) investigates the conformational sampling of polysaccharides and proteins. Electron transfer (ET) reaction is one of the most fundamental processes in chemistry and biology. Because of the quantum nature of the processes and the complicated roles of the solvent, calculating the accurate kinetic and dynamic properties of ET reactions is challenging but extremely useful. Based on the Marcus theory for thermal ET in weak coupling limit, we combined the rigorous ab initio quantum mechanical (QM) method and well-established molecular mechanical (MM) force field and developed an approach to directly calculate a key factor that affects the ET kinetics: the redox free energy. A novel reaction order parameter fractional number of electrons (FNE) was used to characterize the ET progress and to drive the QM/MMMD sampling of the nonadiabatic free energy surface. This method was used for two aqueous metal cations, iron and ruthenium in solution, and generated satisfactory results compared to experiments. In order to further reduce the computational cost, a QM/MM-minimum free energy path (MFEP) method is implemented and combined with the FNE in the calculation of redox free energies. The calculation results using QM/MM-MFEP+FNE generated identical results as the direct QM/MM-MD method for the two metal cations, demonstrating the consistency of the two different sampling strategy. Furthermore, this new method was applied to the calculation of organic molecules and enhanced the computational efficiency 15-30 times than the direct QM/MM-MD method, while maintaining high accuracy. Finally, I successfully extended the QM/MM-MFEP+FNE method to a series of redox proteins, azurin and its mutants, and obtained very accurate redox free energy differences with relative error less than 0.1 eV. The new method demonstrated its excellent transferability, reliability and accuracy among various conditions from aqueous solutions to complex protein systems. Therefore, it shows great promises for applications of the studies on redox reactions in biochemistry. In the studies of force-induced conformational transitions of biomolecules, the large time-scale difference from experiments presents the challenge of obtaining convergent sampling for molecular dynamics simulations. To circumvent this fundamental problem, an approach combining the replica-exchange method and umbrella sampling (REM-US) is developed to simulate mechanical stretching of biomolecules under equilibrium conditions. Equilibrium properties of conformational transitions can be obtained directly from simulations without further assumptions. To test the performance, we carried out REM-US simulations of atomic force microscope (AFM) stretching and relaxing measurements on the polysaccharide pustulan, a (1&rarr;6)-&beta;-D-glucan, which undergoes well-characterized rotameric transitions in the backbone bonds. With significantly enhanced sampling convergence and efficiency, the REMUS approach closely reproduced the equilibrium force-extension curves measured in AFM experiments. Consistent with the reversibility in the AFM measurements, the new approach generated identical force-extension curves in both stretching and relaxing simulations, an outcome not reported in previous studies, proving that equilibrium conditions were achieved in the simulations. In addition, simulations of nine different polysaccharides were performed and the conformational transitions were reexamined using the REM-US approach. The new approach demonstrated consistent and reliable performance among various systems. With fully converged samplings and minimized statistical errors, both the agreement and the deviations between the simulation results and the AFM data were clearly presented. REM-US may provide a robust approach to modeling of mechanical stretching on polysaccharides and even nucleic acids. However, the performance of the REM-US in protein systems, especially with explicit solvent model, is limited by the large system size and the complex interactions. Therefore, a Go-like model is employed to simulate the protein folding/unfolding processes controlled by AFM. The simulations exquisitely reproduced the experimental unfolding and refolding force extension relationships and led to the full reconstruction of the vectorial folding pathway of a large polypeptide, the 253-residue consensus ankyrin repeat protein, NI6C. The trajectories obtained in the simulation captured the critical conformational transitions and the rate-limiting nucleation event. Together with the AFM experiments, the coarse-grained simulations revealed the protein folding and unfolding pathways under the mechanical tension.</p> / Dissertation

Submitted by GLAUCO ALMEIDA (glaucoag@gmail.com) on 2016-03-18T14:17:37Z No. of bitstreams: 1 Glauco Garrido Almeida-TESE-REV.pdf: 3028760 bytes, checksum: f9d5fc9929fc4cee05187b360a597c04 (MD5) / Approved for entry into archive by Sandra Manzano de Almeida (smanzano@marilia.unesp.br) on 2016-03-18T17:11:17Z (GMT) No. of bitstreams: 1 almeida_gg_dr_ilha.pdf: 3028760 bytes, checksum: f9d5fc9929fc4cee05187b360a597c04 (MD5) / Made available in DSpace on 2016-03-18T17:11:17Z (GMT). No. of bitstreams: 1 almeida_gg_dr_ilha.pdf: 3028760 bytes, checksum: f9d5fc9929fc4cee05187b360a597c04 (MD5) Previous issue date: 2015-12-11 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Trabalhos experimentais e teóricos desenvolvidos nos últimos anos evidenciaram a capacidade de alcoóis e polialcoóis em mudar características energéticas em proteínas. No entanto, o mecanismo responsável por esse efeito não está totalmente elucidado. Tomando como exemplo o dipeptídeo de alanina, foram realizadas simulações QM/MM em água, etanol e mistura 60-40 % em volume de água-etanol. A molécula de dipeptídeo foi descrita em nível de cálculo quântico MP2/aug-cc-pVDZ. Em solução, apenas confórmeros αR e PPII foram encontrados na população de equilíbrio. A diferença de energia livre em solução αR  PPII é determinada pelo balanço entre energia interna do soluto e energia de interação. Para o dipeptídeo de alanina, qualquer fator que aumente a energia de interação soluto-solvente favorece o aumento da população de αR. Por outro lado fatores que diminuam esse valor, como a adição de etanol, aumentam a população de PPII. Os resultados indicam solvatação preferencial para o sistema, evidenciado pela formação majoritária da primeira camada de solvatação por moléculas de água (na mistura), embora seja possível encontrar moléculas de etanol em pequenas quantidades ao redor do grupo carboxílico da extremidade N-terminal e, cadeias metílicas laterais. Todavia, o comportamento parece não afetar a diferença na estabilidade de equilíbrio conformacional. / Recent papers, both experimental and theoretical, have highlighted the capacity of alcohols and polyalcohol in modifying the energy landscape in proteins. However, the mechanism underlying this effect is not fully elucidated. Taking as a model-system the alanine dipeptide, QM/MM calculations were performed in water, ethanol and solution 60-40% (volume) water-ethanol. The dipeptide molecule was described by MP2/aug-cc-pVDZ level. In solution, only αR and PPII conformers were found in the conformational equilibrium population. The free energy difference in solution αR  PPII is determined by the interplay between internal energy and the interaction energy. It has been found that, for alanine dipeptide, any factor that increases the solute-solvent interaction energy also promotes an increasing on αR stability, moreover factors that decreases this value, such as the addition of ethanol molecules, increases the PPII stability. The results points to a preferential solvation behavior for the system, as evidenced by the composition of the first solvation shell in the mixture mainly populated by water molecules, although it is possible to find small concentration of ethanol molecules around the carboxyl group of the N-terminal end and around methylic side chains. However, this behavior does not seem to affect the differential conformational stability. / CAPES: 99999.014630/2013-03

Made available in DSpace on 2016-06-02T20:34:11Z (GMT). No. of bitstreams: 1 2275.pdf: 3782224 bytes, checksum: 7d2e45951c92f58bd422ef52a5aaddca (MD5) Previous issue date: 2009-02-17 / Universidade Federal de Sao Carlos / Computer simulation methods were used to characterize the structure and molecular properties of natural and synthetic biopolymers in aqueous solution. The polysaccharides chitin and chitosan, and aliphatic polypeptides were studied. The interest on the chitin and chitosan biopolymers is because of their biodegradability, biocompatibility and potential use as pharmaceutical and technological product. Molecular dynamics simulations have been used to characterize the structure and the solubility of the chitins and chitosans in aqueous solution. The simulated systems were composed by solvated chains, and nanoparticles composed by chains packed in a parallel and anti-parallel fashion, with different percentage and distribution of acetyl groups. The 100% acetylated chitin, whether isolated or in the form of &#945;/&#946;-chitin, adopt the 2-fold helix conformation with &#966; and &#968; values similar to those on crystalline state. The ionic strength affects the kinetics, but not the conformational equilibrium. In solution, the intramolecular hydrogen bond HO3(n)···O5(n+1) responsible for the 2-fold helical motif is stabilized by hydrogen bonding to water molecules in a well-defined orientation. On the other hand, chitosan with small percentage and random distribution of acetil groups can adopt five distinct helical motifs and its conformational equilibrium is highly dependent on pH. The hydrogen bond pattern and solvation around the O3 atom of insoluble chitosan (basic pH) are nearly identical to those quantities in chitin. Chitin and chitosan nanoparticles with block distribution of acetyl groups favor the formation of intermolecular hydrogen bonds and hydrophobic interactions, resulting in more stable aggregates. The water mobility and orientation around polysaccharide chain (highly affected by electrostatic forces) is responsible for the aggregation and solubility of the chitin and chitosan biopolymers. Moreover, a sequential QM/MM methodology is used to study the &#945;-helix stability of aliphatic polypeptides in water solution. The understanding of the folding process is one of the greatest challenges of biophysics, and the first step is the understanding of the formation and stabilization of the secondary structure of a polypeptide. The calculated heat of formation and free energy of solvation showed that the size of side chain is directly related to the &#945;-helix stability. The results suggest that the helix-coil transition of a polypeptide is governed by the equilibrium between the energy used in the folding process and the energy released in the solvation process, showing the solvent effect on &#945;-helix stabilization. The validation of the sequential QM/MM methodology showed that this method is suitable to study the helix-coil transition of polypeptides in solution. The methodology is therefore useful to study solvation effects on the properties of compounds with many conformational degrees of freedom. / Neste trabalho, métodos de simulação computacional foram usados para caracterizar a estrutura e propriedades moleculares de biopolímeros naturais e sintéticos em solução aquosa. Os polissacarídeos quitina e quitosana, e polipeptídeos alifáticos foram os biopolímeros estudados. O interesse nos biopolímeros quitina e quitosana é devido à suas biodegradabilidade, biocompatibilidade e potencial uso como produto farmacêutico ou tecnológico. No presente trabalho, simulações por Dinâmica Molecular foram utilizadas para caracterizar a estrutura e a solubilidade de quitinas e quitosanas em solução aquosa. Os sistemas modelados eram compostos por cadeias solvatadas e nanopartículas formadas por cadeias empacotadas paralelamente e de forma antiparalela, com diferentes percentagens e distribuição de grupos acetil. A quitina 100% acetilada, tanto na forma isolada ou na forma de &#945;/&#946;-quitina adota a conformação de hélice 2, com valores de &#966; e &#968; similares aos da sua estrutura cristalina. A força iônica afeta a cinética, mas não o equilíbrio conformacional. Em solução, as ligações de hidrogênio intramolecular HO3(n)···O5(n+1), responsável por estabilizar o motivo helicoidal hélice 2, são estabilizadas por ligações de hidrogênio com moléculas de água em orientações bem definidas. Por outro lado, a quitosana com pequena percentagem e distribuição randômica de grupos acetil pode adotar cinco motivos estruturais e seu equilíbrio conformacional é altamente dependente do pH. O padrão de ligação de hidrogênio e a solvatação ao redor do átomo O3 da quitina insolúvel (pH básico) é quase idêntico ao observado para a quitina. As nanopartículas de quitina e quitosana com distribuição em blocos de grupos acetil favorece a formação de ligações de hidrogênio intermolecular e interações hidrofóbicas, resultando em agregados mais estáveis. A mobilidade e a orientação das moléculas de água ao redor da cadeia de polissacarídeo (altamente afetada por forças eletrostáticas) é responsável pela agregação e solubilidade dos biopolímeros quitina e quitosana. Além disso, a metodologia QM/MM sequencial foi utilizada para estudar a estabilidade da &#945;-hélice de polipeptídeos alifáticos em solução. Sabe-se que o entendimento do processo de enovelamento é um dos grandes desafios da biofísica, e o primeiro passo consiste em entender a formação e a estabilização da estrutura de polipeptídeos. Os valores de calor de formação e energia livre de solvatação mostraram que o tamanho da cadeia lateral é diretamente proporcional à estabilidade da &#945;-hélice. Os resultados sugerem que o processo de enovelamento-desenovelamento de polipeptídeos é governado pelo equilíbrio entre a energia utilizada para enovelar o peptídeo e a energia liberada pelo processo de solvatação, mostrando o efeito do solvente na estabilização da &#945;-hélice. A validação da metodologia QM/MM sequencial utilizada mostrou ser adequada para o estudo do processo de enovelamento desenovelamento de polipeptídeos em solução, e útil no estudo da estrutura eletrônica e do efeito do solvente em compostos que possuam elevado grau de liberdade conformacional.

Chloroperoxidase (CPO) is a potential biocatalyst for use in asymmetric synthesis. The mechanisms of CPO catalysis are therefore of interest. The halogenation reaction, one of several chemical reactions that CPO catalyzes, is not fully understood and is the subject of this dissertation. The mechanism by which CPO catalyzes halogenation is disputed. It has been postulated that halogenation of substrates occurs at the active site. Alternatively, it has been proposed that hypochlorous acid, produced at the active site via oxidation of chloride, is released prior to reaction, so that halogenation occurs in solution. The free-solution mechanism is supported by the observation that halogenation of most substrates often occurs non-stereospecifically. On the other hand, the enzyme-bound mechanism is supported by the observation that some large substrates undergo halogenation stereospecifically. The major purpose of this research is to compare chlorination of the substrate beta-cyclopentanedione in the two environments. One study was of the reaction with limited hydration because such a level of hydration is typical of the active site. For this work, a purely quantum mechanical approach was used. To model the aqueous environment, the limited hydration environment approach is not appropriate. Instead, reaction precursor conformations were obtained from a solvated molecular dynamics simulation, and reaction of potentially reactive molecular encounters was modeled with a hybrid quantum mechanical/molecular mechanical approach. Extensive work developing parameters for small molecules was pre-requisite for the molecular dynamics simulation. It is observed that a limited and optimized (active-site-like) hydration environment leads to a lower energetic barrier than the fully solvated model representative of the aqueous environment at room temperature, suggesting that the stable water network near the active site is likely to facilitate the chlorination mechanism. The influence of the solvent environment on the reaction barrier is critical. It is observed that stabilization of the catalytic water by other solvent molecules lowers the barrier for keto-enol tautomerization. Placement of water molecules is more important than the number of water molecules in such studies. The fully-solvated model demonstrates that reaction proceeds when the instantaneous dynamical water environment is close to optimal for stabilizing the transition state.

No description available.

Durant cette thèse, nous avons abordé l'étude de la réactivité en phase gazeuse des biomolécules. L’avènement des techniques d’ionisation douces telle que l’ionisation par éléctronébulisation, a rendu possible ces dernières années, la formation d'ions en phase gazeuse sans dégrader la biomolécule étudiée.La Dissociation Induite par Collision (CID) est un cas particulier de spectrométrie de masse en tandem, que nous avons utilisée durant ce travail. Le principe du CID est d'activer les modes rovibrationnelles d’un système moléculaire ionique par collision avec un gaz inerte, ce qui augmente la probabilité de fragmentation de l'ion. Bien qu'étant une technique très utile d'un point de vue analytique, la spectrométrie de masse en tandem ne donne pas d'informations sur les mécanismes des réactions se produisant dans la cellule de collision; afin d’obtenir ces informations, les simulations de dynamique chimiques apparaissent comme un outil satisfaisant. En effet, en utilisant la dynamique directe, nous évitons ainsi d'explorer la totalité de la surface d'énergie potentielle, qui devient compliquée lors de l’étude d’édifices moléculaires de grande taille. Etant donné que les simulations de dynamique chimiques sont limitées à de courtes échelles, de l’ordre de la dizaine de picosecondes, nous avons également employé la théorie unimoléculaire RRKM (Rice-Ramsperger-Kassel-Marcus) pour étudier la réactivité à des temps plus longs, en vue de comprendre les processus réactionnels se produisant à l’issue du processus de relaxation vibrationnelle intramoléculaire (IVR). Durant ce travail de thèse, nous avons choisi d'étudier comme système modèle de base nucléique la molécule d'uracile. Par ailleurs,nous avons aussi étudié la réactivité en phase gazeuse de sucres (cellobiose, maltose et gentiobiose), qui ont été au préalable dérivatisés afin de localiser la charge sur la molécule et ainsi simplifier l’étude théorique associée. / In the present thesis, we address the study of the reactivity of biomolecules in the gasphase.The advent of soft ionization techniques such as electrospray ionization, made possible, in the last years, the gentle formation of ions in the gas phase without breaking the molecule understudy.Collision Induced Dissociation (CID) is aparticular case of tandem mass spectrometrydynamics simulations are pointed like asatisfactory tool. Using direct dynamics weavoid exploring the whole potential energysurface, which becomes really complicatedwhen dealing with big molecules.Since chemical dynamics simulations arerestricted to the short time scale reactivity,typically ~10ps, we make use of the Rice–Ramsperger–Kassel–Marcus (RRKM)unimolecular theory to study the reactivity atUniversité Paris-SaclayEspace Technologique / Immeuble DiscoveryRoute de l’Orme aux Merisiers RD 128 / 91190 Saint-Aubin, Francethat we use in the present thesis. The aim of CIDis to activate the rovibrational modes of an ionicmolecular system by collisions with an inert gas,increasing the probability of the ion of beingfragmented.Despite being a really useful technique, tandemmass spectrometry does not give informationabout the mechanisms of the reactions takingplace in the collision cell; in order to obtain suchinformation, chemicallonger time scales to understand reaction pathsthat take place after intramolecular vibrationrelaxation (IVR).In the present thesis we have chosen to study asmodel system of nucleobase the uracil molecule.Furthermore, we also studied the gas-phase reactivity of carbohydrates (cellobiose, maltose and gentiobiose), which were preliminarily derivatized in order to simplify the charge localization, and consequently the theoretical study.

京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第19521号 / 理博第4181号 / 新制||理||1600(附属図書館) / 32557 / 京都大学大学院理学研究科化学専攻 / (主査)教授 林 重彦, 教授 谷村 吉隆, 教授 松本 吉泰 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

In this work, the development of parameters for a series of imidazolium-based ionic liquids molecules, now included in the AMOEBA force field, is discussed. The quality of obtained parameters is tested in a variety of calculations to reproduce structural, thermodynamic, and transport properties. First, it is proposed a novel method to parameterize in a faster, and more efficient way parameters for the AMOEBA force field that can be applied to any imidazolim-based cation. Second, AMOEBA-IL polarizable force field is applied to study the N-tert-butyloxycarbonylation of aniline reaction mechanism in water/[EMIM][BF4] solvent via QM/MM approach and compared with the reaction carried out in gas-phase and implicit solvent media. Third, AMOEBA-IL force field is applied in alchemical calculations. Free energies of solvation for selected solutes solvated in [EMIm][OTf] are calculated via BAR method implemented in TINKER considering the effect of polarization as well as the methodology to perform the sampling of the alchemical process. Finally, QM/MM calculations using AMOEBA to get more insights into the catalytic reaction mechanism of horseradish peroxidase enzyme, particularly the structures involved in the transition from Cp I to Cp II.